Liquid-microjet photoelectron spectroscopy of the green fluorescent protein chromophore

Abstract

Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. However, the most fundamental property underpinning photooxidation, the electron detachment energy, has only been measured for the deprotonated GFP chromophore in the gas phase. Here, we use multiphoton ultraviolet photoelectron spectroscopy in a liquid-microjet and high-level quantum chemistry calculations to determine the electron detachment energy of the GFP chromophore in aqueous solution. The aqueous environment is found to raise the detachment energy by around 4 eV compared to the gas phase, similar to calculations of the chromophore in its native protein environment. In most cases, electron detachment is found to occur resonantly through electronically excited states of the chromophore, highlighting their importance in photo-induced electron transfer processes in the condensed phase. Our results suggest that the photooxidation properties of the GFP chromophore in an aqueous environment will be similar to those in the protein.

Accurate Vertical Ionization Energy of Water and Retrieval of True Ultraviolet Photoelectron Spectra of Aqueous Solutions

“…This observation is consistent with the results of our recent photoelectron spectroscopy study of the green fluorescent protein chromophore in aqueous solution, in which one-dimensional electron scattering simulations suggested that the eKE loss was <0.2 eV. 14 …”

“…Liquid-jet PES (LJ-PES) is now an active research field involving a growing number of research groups around the world. 4 − 14 Nonetheless, the requirement for relatively high concentrations of solute to obtain adequate signal-to-noise ratio after subtracting the photoelectron spectrum of water has excluded most X-ray LJ-PES studies of aqueous solutions of organic molecules because organic molecules tend to be only weakly soluble. 9 A solution to this problem is to use resonance-enhanced PES with ultraviolet (UV) light pulses.…”

“… 9 A solution to this problem is to use resonance-enhanced PES with ultraviolet (UV) light pulses. 9 , 14 − 23 Unfortunately, a major challenge that has been hampering the development of UV LJ-PES for aqueous solutions is a lack of consensus on the precise effect of inelastic scattering of low kinetic energy electrons in liquid water on peak shapes and positions. 24 − 30 …”

“…It is also worth noting that neither of these methods considered, explicitly, non-uniform solute concentration depth profiles, which are common for weakly soluble organic molecules that tend to have an enhanced surface concentration. 14 , 36 , 37 …”

“…For a long time, PES was restricted to the gas or solid phases, due to the requirement for high vacuum to minimize scattering of the emitted electrons. , However, the introduction of liquid jets and their combination with intense X-ray sources at synchrotrons, in the late 1990s, expanded the scope of PES to include liquids. Liquid-jet PES (LJ-PES) is now an active research field involving a growing number of research groups around the world. − Nonetheless, the requirement for relatively high concentrations of solute to obtain adequate signal-to-noise ratio after subtracting the photoelectron spectrum of water has excluded most X-ray LJ-PES studies of aqueous solutions of organic molecules because organic molecules tend to be only weakly soluble . A solution to this problem is to use resonance-enhanced PES with ultraviolet (UV) light pulses. ,− Unfortunately, a major challenge that has been hampering the development of UV LJ-PES for aqueous solutions is a lack of consensus on the precise effect of inelastic scattering of low kinetic energy electrons in liquid water on peak shapes and positions. − …”